Intramolecular shielding maintains the ER Ca sensor stim1 in an inactive conformation

Store-operated calcium entry (SOCE) represents a major calcium influx pathway in non-excitable cells and is central to many physiological processes such as T cell activationand mast cell degranulation. SOCE is activated through intricate coordination between theCa sensor on the ER membrane (stromal interaction molecule 1, STIM1) and the plasma membrane channel Orai1. When Ca stores are depleted, STIM1 oligomerizes and physically interacts with Orai1 through its SOAR/CAD domain, resulting in Orai1 gating and Ca influx. Here, we describe novel inter- and intramolecular FRET sensors in the context of the full-length membrane-anchored STIM1, and show that STIM1 undergoes a conformational change in response to store depletion to adopt a stretched 'open' conformation that exposes SOAR/CAD and allows it to interact with Orai1. Mutational analyses reveal that electrostatic interactions between the predicted first and third coiled-coil domains of STIM1 are not involved in maintaining the 'closed' inactive conformation. In addition, the results argue that an amphipathic a-helix between residues 317 and 336 in the so-called inhibitory domain is important to maintain STIM1 in a closed conformation at rest. Indeed, mutations that alter the amphipathic properties of this helix result in a STIM1 variant that is unable to respond to store depletion in terms of forming puncta, translocation to the cortical ER or activating Orai1. © 2013. Published by The Company of Biologists Ltd.